A major challenge in the post-genomic era of biomedicine is to define the functions of newly identified gene products in normal and pathological processes. Traditional molecular genetics techniques are being employed in large-scale efforts to determine the functions of these gene products. However, these techniques are often inadequate when gene products are essential and for organisms that are difficult to genetically manipulate, such as primary human cells or the malarial parasite. To help address this challenge, we are developing a chemical genetics platform for discovery and functional analysis of peptidases. In this proposal, we first describe the synthesis and validation of novel libraries of activity-based probes (ABPs) that are specific for metallo-aminopeptidases (MAPs). MAPs are zinc-dependent enzymes that catalyze the hydrolysis of one or two amino acids adjacent to the amino terminus from a protein or peptide. They have essential roles as both digestive and regulatory enzymes in many organisms, including humans and pathogens. In the second part of this proposal we will apply these ABPs to study MAP function in the human malaria parasite, Plasmodium falciparum. Malaria is a global disease causing 500 million clinical cases and more than 1 million deaths each year. Drug resistance in P.falciparum, the organism that causes most malaria associated deaths, has now become a major problem necessitating the discovery of novel antimalarials. Peptidases participate in several critical pathways during the life cycle of P. falciparum, and aminopeptidases are hypothesized to assist in the degradation of host hemoglobin. However, of the four MAPs in the P. falciparum genome, three are genetically essential and current genetic tools in this parasite do not allow for function analysis. Therefore, we will develop the first specific ABPs for each of these parasitic aminopeptidases and will apply them to directly address the function of these essential enzymes in live parasites. PUBLIC HEALTH RELEVANCE: A major challenge in post-genomic era of biomedicine is to define the functions of newly identified gene products in normal and pathological processes. We are developing a chemical genetics platform for peptidase discovery and functional analysis. In this proposal, we describe the development of small molecule metallo-aminopeptidase activity-based probes and their application in elucidating metallo-aminopeptidase function in the human malaria parasite Plasmodium falciparum. Perhaps the greatest impact of this proposal will be to provide a suite of tools and techniques that will be able to study aminopeptidase mediated pathways in a wide range of biological systems.